Sensitized genetic systems and DNA at-risk motif reporters have been developed through several years of investigating genetic risks and synergistic interactions with DNA metabolic defects. These two methodologies allow us to identify the strongest cases of genome instability and to understand molecular mechanisms underlying the instability. Repeat motifs have been identified in many organisms that are at high risk for genetic change in wild-type or mutation-prone cells. They can be a source of genomic instability, cancer and in some cases provide changes that extend the host range of infectious microbes. We surmised from our various experimental approaches that many of these At Risk Motifs (ARMs) can form non-canonical DNA structures that are poor substrates for replication or post-replication repair. This led us to propose that ARMs can be a major source of genome instability. Once the underlying mechanisms are unraveled, ARMs also provide a powerful tool of discovery in mechanistic studies of DNA metabolism. Using a variety of genetic and molecular approaches with yeast, we have characterized several types of ARMs and employed them to address mechanisms of genome instability. These include small direct repeats separated by up to 100 bases, a motif common in all organisms, long inverted repeats in configurations similar to many arrangements of the frequently occuring Alu and LINE sequences in the human genome, homonucleotide runs as short as 8 to 10 bases that can lead to extremely high levels of frameshift mutation and other types of unstable mini- and microsatellites. We have established that defects in several aspects of DNA metabolic processes greatly exacerbate the instability associated with ARMs, in essence dramatically increasing the risk associated with the at-risk motifs. DNA repair, replication and processing of DNA intermediates require coordinated interactions between many proteins. The combination of subtle changes in one or more DNA metabolic acting at ARMs can lead to synergistic increases in genome instability. We employ a variety of sophisticated genetic and biochemical approaches to identify combinations of genes that are important for maintaining genome stability. Our studies over the last several years have concentrated on the interplay between genes involved in DNA replication, double-strand break repair, mismatch repair and base excision repair in maintaining genome stability, particularly at ARM sites. We concentrated our recent research on the multiple biological roles of the Pol delta-exonuclease. For over thirty years, proofreading of errors that arise during replication was considered the only biological function of this enzymatic activity. A defect in proofreading can increase the mutation rate and lead to synthetic lethality or extreme hypermutability when combined with a defect in postreplication mismatch repair (MMR). Based on specific genetic interactions or proofreading mutants with defects in Exo1 and Rad27/Fen. along with biochemical analysis of yeast Pol delta we have discerned additional biological roles for the intrinsic exonuclease of Pol delta, specifically in the maturation of Okazaki fragments and in MMR. We have questioned whether the Pol delta-Exo performs all these biological functions--proofreading, Okazaki maturation, and MMR--in association with the replicative complex or as an exonuclease separate from DNA replication. We identified a novel category of yeast Pol delta mutants at amino acid 523, Pol3-L523X, that are defective in processive DNA synthesis in the presence of PCNA, but only when the error rate of incorporation is high because of a dNTP imbalance. Surprisingly, the mutant enzymes retained robust 3',5'-exonuclease activity. Based on their biochemical properties, the mutant holoenzymes appear to be impaired in switching of the nascent 3'-end of the newly synthesized DNA strand between the polymerase and the exonucleaseactive sites. Based on our previous studies we identified a set of characteristic in vivo genetic features that indicate strongly implicate the Pol delta in mismatch repair and in Okazaki maturation in lagging strand DNA replication. These features included synergistic interaction with defects in 5'-exonuclease EXO1 and in 5'-flap endonuclease RAD27/FEN1, as well as highly increased mutation rates of duplications flanked by short dispersed repeat ARMs. All the features of the pol3-L523X mutants, including mutation rates and spectra as well as negative synergy with defects in msh2, exo1 and rad27/fen1,were indistinguishable from previously studied Exo-defective mutants. Thus, all three biological functions of Pol delta-Exo appear to be severely impaired by the switching defect in Pol3-L523X mutations. We have concluded that all three biological functions are performed by Pol delta-Exo within the replication complex during DNA replication.